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Culture System to Blastocyst

Culture System to Blastocyst. Başak Balaban, BSc American Hospital of Istanbul. AMERICAN HOSPITAL. 6 Mart 2010 Resmi gazete sayı: 275513 Sağlık Bakanlığı: ÜREMEYE YARDIMCI TEDAVİ UYGULAMALARI VE ÜREMEYE YARDIMCI TEDAVİ MERKEZLERİ HAKKINDA YÖNETMELİK. ALTINCI BÖLÜM

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Culture System to Blastocyst

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  1. Culture System to Blastocyst Başak Balaban, BSc American Hospital of Istanbul AMERICAN HOSPITAL

  2. 6 Mart 2010 Resmi gazete sayı: 275513 Sağlık Bakanlığı: ÜREMEYE YARDIMCI TEDAVİ UYGULAMALARI VE ÜREMEYE YARDIMCI TEDAVİ MERKEZLERİ HAKKINDA YÖNETMELİK ALTINCI BÖLÜM Yasaklar ve Sorumluluklar ile Denetim ve İdarî Müeyyide ÜYTE uygulamaları ile ilgili yasaklar              MADDE 18 (8)   b) Merkezlerde ÜYTE uygulamasında birden fazla embriyo transfer edilmemesi esastır. Ancak, 35 yaşa kadar birinci ve ikinci uygulamada tek embriyo, üçüncü ve sonraki uygulamalarda iki embriyo, 35 yaş ve üzerinde tüm uygulamalarda en fazla iki embriyo transfer edilebilir.

  3. Embryo quality&Implantation • Transfer of good quality embryos results in higher pregnancy and implantation rates • Highest implantation rate with the minimum number of embryos transferred (SET) should be our final goal • Therefore selection of the TOP QUALITY EMBRYO/S for transfer is an essential process that should be achieved in IVF lab.s Hsu F&S 1999, Terriou F&S 2001, De Neubourg HR 2004, Schmidt 2005

  4. Markers determining embryo quality&viability • Embryological/morphological markers • Molecular markers(chromosomal integrity&expression of the appropriate developmental genes;cumulus granulosa cell) • Metabolic analysis of the embryo, Metabolomics, Proteomics........ • Biochemical markers (reactive oxygen species) • Non-invasive techniques to select the most viable oocyte(miotic spindle/ZP birefrigence) and sperm (IMSI, PICSI...)before the embryo development

  5. Sperm BLASTOCYST IN VITRO CULTURE OOCYTE Early Cleavage Quality Isiklar&Balaban J Rep.Med 2002 Balaban FS 2000, Gardner FS 2000 PN Morphology Day 2 embryo Day 3 embryo Balaban HR 2001 Rjinders HR 1998, Shapiro FS 2000,Ragione R.B.Endocrinol 2007 Multinucleation Yakin&Balaban FS 2006 0 16-18 24-25 48 72 96-120

  6. Blastocyst formation rates Blake et al.,Cochrane Review 2007

  7. Why an alternative for cleavage stage embryo transfer?ADVANTAGES • 1. The embryo is transversing the fallopian tube at cleavage stage; it’s in the uterus at blastocyst stage, so premature exposure of early stage embryos to the uterine environment may cause homeostatic stress on the embryo, resulting in a reduced implantation potential(Gardner 1996) • 2. Selection of only the embryos that have demonstated the potential for continued development under embryonic genomic control (Braude 1988) • 3. If a blastocysts is more viable than a cleavage-stage embryo than BT could result in higher IR, which gives the possibility of transferring fewer embryos that lowers the costly multiple birth rates (Jones 1999) Johnson et al.,Best Practice&Research Clin.Obst.Gyanec. 2007

  8. Critics of blastocyst culture • 1.Having no embryos to transfer(Marek 1999) (The day of patient recruitment into the BT is crucial to this argument) • 2. Failure to have extra embryos that can be freeze-stored for future use(Tsirgotis 1998)(Few articles published reporting the impact on the final outcome of PR&LBR per started cycle) • 3. MZ twinning( Only retrospective studies were able to show an increased frequency,Behr 2000, Da Costa 2001, Jain 2004) • 4. Altered sex ratio in births(Menezo 1999, Hentemann 2009,all retrospective studies??) • 5. Sensitivity of the system to suboptimal conditions Johnson et al.,Best Practice&Research Clin.Obst.Gyanec. 2007

  9. Blastocyst Grading • BG1 early cavitation resulting in an eccentric and then expanded cavity lined by a distinct ICM region and TE layer • BG2 delayed initial cavitation exhibiting a transitional phase between early cavitation and expansion • BG3 blastocysts with several degenative foci in the ICM; cells appear dark and necrotic Docras, Hum Reprod 1993

  10. Blastocyst quality BG1 BG3 BG2 Docras Grading System

  11. Gardner & Lane Fertil Steril 2000 1. 2. Early blastocyst: blastocoel < half the volume of the embryo Blastocyst: blastocoel > half the volume of the embryo 5.Hatching 3. 4. 6.Hatched Full blastocyst: blastocoel Completely fills the embryo Expanded blastocyst: blastocoel volume is now larger than that of the early embryo and the zona is thinning

  12. Scoring System for Human Blastocyst ICM Grading A) Tightly packed, many cells B) Loosely grouped, several cells C) Very few cells Trophectoderm Grading A) Many cells forming a cohesive epithelium B) Few cells forming a loose epithelium

  13. Blastocyst grading 2 x 4AA Good quality ICM Good quality trophoectoderm 5BB

  14. Outcome of homogenous blastocyst transfers Balaban et al., F&S, 2000

  15. Effect of blastocyst score on pregnancy *p<.001,**p<.01 Gardner et al. Fertil Steril 2000

  16. Comparison of two blastocyst grading systems Balaban et al.,F&S 2006

  17. ALPHA & ESHRE SIGE CONCENSUS WORKSHOP ON EMBRYO GRADING Concept:2 day meeting of experts to present, discuss and agree on key aspects required for embryo selection/grading. The experts were asked to join actively with the expectation thatall will agree and endorse a final grading scheme at the conclusion of our meeting. Venue: Istanbul Date: February 26th-27th. 2010 Participants:Başak Balaban, Daniel Brison, Gloria Calderón, James Catt, Joe Conaghan, Lisa Cowan, Thomas Ebner, David Gardner, Thorir Hardarson, Kersti Lundin, Cristina Magli, David Mortimer, Sharon Mortimer, Santiago Munné, Dominique Royere, Lynette Scott, Johan Smitz, Alan Thornhill, Jonathan van Blerkom, Etienne Van den Abbeel **Submitted to HR & RBM Online

  18. Blastocyst culture:Is BT more successful than ET?? • Retrospective studies in selected groups. • Retrospective studies in all IVF patients. • Prospective randomised trials • * Selected groups • * All IVF patients

  19. Cleavage Stage versus Blastocyst stage embryo transfer in ARTBlake DA, Farquar CM, Johnson N, Proctor MCochrane Database of Systematic Reviews 2007 CD002118 ***18 RCT comparing early stage ET (Day 2 to 3) with BT (Day 5 to 6) were included ( 14-published articles, 4 abstracts), women aged <40 years, The mean age varied from 29 to 34 • Primary outcome: LBR per couple (no.of live-births per couple) • Secondary outcome: CPR, MPR, high order MPR, miscarriage, embryo freezing, failure to have any ET rate per couple • Outcomes not appropriate for statistical pooling: Live births per OPU and ET,CPR per OPU and ET, implantation rate

  20. Cleavage Stage versus Blastocyst stage embryo transfer in ART Cochrane Review 2007

  21. Live birth rate per couple: Cleavage&Blastocyst transfer Blake et al.,Cochrane Review, 2007

  22. Failure to transfer any embryos per couple Blake et al.,Cochrane Review, 2007

  23. Embryo freezing per couple Blake et al.,Cochrane Review, 2007

  24. Cumulative pregnancy rates from fresh&frozen ET Blake et al.,Cochrane Review, 2007

  25. Early PR loss is sig. higher after day 3 single ET than day 5 single BT Patients < 36, ≤2rank trial, Equal no.of ET in each group, GnRH antogonist protocol Papanikolaou et al.,RBM Online 2006

  26. Single ET& BT: A prospective randomised trial Patients ≤36, 1st./2nd. Attempt, ≥5 2PN PR: 42.2% when ≥4G1, with 95.5% suitable for SBT, PR: 27.8%when ≤3G1, with 88.5% suitable for SBT Zech et al.,F&S 2007

  27. Randomization on a voluntary basis, SBT sig.younger (31.9 vs. 32.9), no dif. for other clinical parameters, selection regardless of the no.or quality on day 2 Mean no.of MII: 7.0-7.5 Mean no.of emb: 5.1-5.3 Guerif et al., HR 2009

  28. FETs: Higher (not.sig.) cryosurvival with embryos than with blastocysts higher no.of deliviries(not. sig..) with sET compared with the SBT Guerif et al., HR 2009

  29. Blastocyst transfer for unselected & poor response patient population (Cochrane review 2007 Prospective Randomized trials ****Unselected population • Karaki 2002: No: 162 , Patients with ≥5 2PN, sig. higher IR with day 5 vs. day 3 (13% vs. 26%) BUT sig. higher cancellation rate(CR) with day 5 (0 vs. 11%) • Van der Auwera 2002: No: 129, unselected population, sig.higher CPR, LBR, IR for day 5 when compared with day 2 BUT sig. higher CR for day 5 • Emiliani 2003: No: 171, ≥4 2PN, Day 2 vs. day 5- higher CR, similar IR, lower delivery rate(DR), and cumulative DR • Kolibianakis 2004 :No: 442, all cases <43 age, sig.higher CR with day 5 vs. day 3, lower no. of cryo cycles with day 5, but similar OPR and IR ****Poor prognosis patients • Levitas 2004: No:54, <37 age,≥3 RIF, higher CR (26%) with day 5 vs. day 3, BUT sig. higher IR with day 5

  30. Prospective quasi-randomized, mixed general IVF population, No sig.dif. clinical parameters Weissman et al., RBM Online 2008

  31. Cancellation rate: 12/70: 17.1% *** Weissman et al., RBM Online 2008

  32. Total:587 Natural conception:0.42% Demographics&Etiology : no stat.dif. Except mean age(29.9Bvs 30.8 E.) Treatment charecteristics: no.stat.dif. Except no.of COCs (12.3B vs. 10.4E) **Delivery of 24 healthy babies reported MZT: Hardening of ZP, disorder in the cell adhesion process secondary to the culture media, Costa HR 2001, Milki F&S 2003) ICSI causing splitting of ICM through the artifical gap (Tarlatzis F&S 2002). Papanikolaou et al.,F&S 2010

  33. Kallen et al., F&S 2010

  34. Kallen et al., F&S 2010

  35. Retrospective population-based study of 150.376 autologous ETs in Australia 2002-2006 >1/5 of the fresh BT results in an healty baby!, sig. better perinatal outcomes when compared with cleavage ET FET. More healthy babies when frozen at cleavage, and cultured till blastocyst after thawing Wang et al.,HR 2010 Wang et al., HR 2010 sBT optimizes the chance of a healthy Term baby when compared with sET, 34.035 cycles

  36. CONCLUSION Culture System to Blastocyst: Is it clinically more successful than cleavage stage ET applications?? • Blastocyst transfer results in sig. higher LBRs when compared with cleavage stage ETs. for good prognosis patients. The success of BT is dependant on the no.of good quality 8 embryos on day 3 (≥ 3 is suggested) • Cleavage stage embryo transfer should be preferred for poor prognosis patients (very high cancellation rates are reported), it may also be suggested for cases with <3 good quality 8 cell embryos • Neonatal safety of BTs compared to ETs. still remains to be investigated

  37. Papanikolaou et al.,F&S 2010

  38. Kallen et al., F&S 2010

  39. Kallen et al., F&S 2010

  40. Guerif et al., HR 2009

  41. Similar delivery rates in a selected group of patients, for day2&5 embryos cultured in sequential medium Patients with <4 IF,<39, ≥4 2PN, B formation rate:48.3%, Emiliani et al.,HR 2007

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